Peptides have been synthesized by condensation of amino acids. The peptide synthesis methods fall into two general categories; liquid-phase and solid-phase. The liquid-phase method has disadvantages, resulting from use of a solution, of being highly time consuming and labor intensive before it is completed, because the intermediate must be extracted from the solution, refined and confirmed whenever amino acid is added to, and chain length elongation is conducted in the solution. The solid-phase method elongates the peptide chain by a simple procedure in which an activated amino acid derivative is condensed consecutively on a resin on which amino acid with the protected amino terminals is and removing the protective group for the amino terminals. It is a simple peptide-synthesis method, much more efficient than the liquid-phase method.
One of the solid-phase methods uses t-butoxycarbonyl amino acid (hereinafter "Boc-amino acid"). It immobilizes Boc-amino acid on a resin, and condenses activated Boc-amino acid, while removing the Boc groups as the protective group for the amino terminals. It needs a large quantity of a halogen-containing compound, such as trifluoroacetic acid and methylene chloride, to remove the Boc group, and also needs a strong acid, such as anhydrous hydrogen fluoride to take peptide out of the resin in the final stage. A peptide thiol ester can be easily synthesized by the aid of Boc-amino acid. Nevertheless, however, this method involves environmental and safety-related problems, resulting from use of a halogen-containing compound and strong acid.
Another solid-phase method uses fluoren-9-ylmethoxycarbonyl amino acid (hereinafter "Fmoc-amino acid"). It condenses Fmoc-amino acid, while removing the Fmoc groups as the protective group for the amino groups with the aid of an amine compound, such as piperidine. It has been widely used, because of its advantages of dispensing with a halogen-containing compound for elongating the peptide chains and a strong acid for taking peptide out of the resin. This method, although preferable in terms of environment and safety, has an inherent disadvantage, when applied to synthesis of peptide thiol ester: peptide thiol ester shows high reactivity with the amine, decomposing the thiol ester bond to prevent and vitiate peptide chain elongation.
It is an object of the present invention to provide a method for producing a peptide thiol ester relatively easily, while reducing problems related to environmental preservation and safety. It is highly desirable to provide a synthetic scheme which does not require halogen containing compounds such as methylene chloride and which does not require reagents which reduce yields by reacting with desired intermediates. Such a method is very useful for synthesis of peptides, such as a long-chain peptides and cyclopeptides, and, at the same time, can be widely used for a variety of medicines from peptides as the starting stocks.